The overall objective of the proposed project is to characterize and localize the acid phosphatases in developing bones and the enamel organ and pulp of tooth buds and to determine the effect of parathyroid hormone and vitamin D in vivo/in vitro on the different phosphatases, in order to gain insight into the role of these enzymes in hard tissue formation and function. The acid phosphatases in rat tissue homogenates have been separated and purified to differnet degrees by column chromatography. Characterization of the enzymes will be completed with regard to molecular size, pH optimum, thermolability, kinetic characteristics for various substrates and the influence of various effectors on the kinetic charactristics. Information concerning the function of the various acid phosphatases will be obtained by determining (a) the specificity of the enzymes for potential in vivo substrates; (b) localization of enzymes in cells and cellular organelles and (c) by determining the effect of vitamin D and parathyroid hormone on activity and tissue level of specifici acid phosphatases in bone and tooth buds in vivo and in bone cultured in vitro. The latter studies will be carried out with vitamin D-deficient suckling rats, or, for the in vitro experiments, with bones or isolated bone cells from such rats. Localization of the enzymes will be determined by means of (a) biochemical studies on dissected tissues containing primarily one type of cell; (b) cytochemical techniques at the light and electron microscopic levels using specific substrates and inhibitors determined biochemically; and (c) immunocytochemical techniques using antiserum to one or more specific phosphatases.